Abstract
The antimicrobial peptide LL-37 inhibits the growth of the major human pathogen Mycobacterium tuberculosis (Mtb), but the mechanism of the peptide–pathogen interaction inside human macrophages remains unclear. Super-resolution imaging techniques provide a novel opportunity to visualize these interactions on a molecular level. Here, we adapt the super-resolution technique of stimulated emission depletion (STED) microscopy to study the uptake, intracellular localization and interaction of LL-37 with macrophages and virulent Mtb. We demonstrate that LL-37 is internalized by both uninfected and Mtb infected primary human macrophages. The peptide localizes in the membrane of early endosomes and lysosomes, the compartment in which mycobacteria reside. Functionally, LL-37 disrupts the cell wall of intra- and extracellular Mtb, resulting in the killing of the pathogen. In conclusion, we introduce STED microscopy as an innovative and informative tool for studying host–pathogen–peptide interactions, clearly extending the possibilities of conventional confocal microscopy.
Highlights
Despite the availability of effective antibiotic treatment of tuberculosis, the management of this devastating disease remains a major challenge
We evaluated the suitability of innovative stimulated emission depletion (STED) microscopy as a tool for directly observing host–pathogen interaction using, as an example, LL-37, primary human macrophages and virulent Mycobacterium tuberculosis (Mtb)
In STED imaging performed with the same instrument as used for confocal microscopy, the identical bacterium shows a distinct
Summary
Despite the availability of effective antibiotic treatment of tuberculosis, the management of this devastating disease remains a major challenge. Cathelicidin is a cationic antimicrobial peptide (hCAP18) that is up-regulated in human macrophages by a vitamin D-dependent pathway [4]. It is cleaved into the active form LL-37, which is active against extra- and intracellular Mycobacterium tuberculosis (Mtb). Other possibilities include the impairment of cellular processes and pore formation caused by ionic interactions of the peptide and the bacterial cell wall [3,8,9]. LL-37 enters phagosomes where intracellular bacteria are either directly eliminated by bacterial cell wall degradation or indirectly by inducing the cellular process of degradation with lysosomal fusion [10]. We evaluated the suitability of innovative STED microscopy as a tool for directly observing host–pathogen interaction using, as an example, LL-37, primary human macrophages and virulent Mtb
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